Tissue staining method, tissue evaluation method and biosubstance detection method

ABSTRACT

A tissue staining method which comprises: staining a tissue with a staining reagent wherein a biosubstance recognition site is bonded to particles carrying multiple fluorescent substances accumulated therein; in the stained tissue, counting fluorescent points or measuring fluorescent brightness; and evaluating the expression level of a biosubstance, which matches the biosubstance recognition site, in the aforesaid tissue on the basis of the number of the fluorescent points or fluorescent brightness that was measured.

TECHNICAL FIELD

The present invention relates to a tissue staining method, a tissueevaluation method, and a biosubstance detection method.

BACKGROUND ART

In pathological diagnosis, first, a sampled tissue is dehydrated andblocked with paraffin to fix the tissue, then the sample is cut intosections haying a thickness of 2 to 8 μm followed by removal of theparaffin therefrom, and subsequently the sections are stained to performmicroscopic observation. A pathologist performs diagnosis on the basisof morphological information and staining information such as changes insize and shape of cell nuclei and changes in tissue pattern in themicroscopic image. The development of image-digitalizing technology hasalso facilitated wide use of automated pathological diagnosis supportequipment that displays information necessary for pathological diagnosisby a pathologist through extraction and measurement of a pathologicalimage input as a digital color image by using a microscope, a digitalcamera, or any other device in the field of pathological diagnosis.

For example, Patent Document 1 discloses a pathological diagnosissupport equipment including a nucleus/cytoplasm distribution-estimatingunit for specifying a cell nucleus region and a cytoplasm region from apathological image; a glandular cavity distribution-extracting unit forspecifying a glandular cavity region (region almost not containing acellular structure) from a pathological image; a cancer site-estimatingunit for determining whether or not cancer cells are present; astage-determining unit for determining the stage of cancer progression;and an image display unit for displaying, for example, a cancer celldistribution map and the stage of progression.

Patent Document 2 discloses a method to detect cancer cells by staininga pathological specimen with two types of dyes selectively andrespectively staining the normal site or the cancer site, evaluating thestaining concentrations from a spectral image in accordance withLambert-Beer's law, and determining whether cancer cells are present.

In each method for evaluation, however, the tissue staining is performedby conventional dye staining (e.g., hematoxylin-eosin staining) or dyestaining using an enzyme (e.g., DAB staining), and the stainingconcentration considerably varies depending on environmental conditionssuch as temperature and time. Accordingly, such pathological diagnosissupport equipments cannot maximize its performance in precisequantitative measurement.

Meanwhile, a fluorescent dye having high sensitivity is also used as alabeling reagent in place of the dye described above in study of tissuestaining (see Non-Patent Document 1). The present inventors observed apathological section prepared with an organic fluorescent dye, FITC,under a fluorescence microscope in accordance with the method ofidentifying/quantitating cells disclosed in Patent Document 3.Unfortunately, the luminescent brightness was too weak to automaticallydetermine a significantly small amount of biomarker on the basis of thelight emission level. Hence, the method requires further improvements.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: Japanese Patent Application Laid-Open Publication    No. 2004-286666-   Patent Document 2: Japanese Patent Application Laid-Open Publication    No. 2001-525580-   Patent Document 3: Japanese Patent Application Laid-Open Publication    No. Sho 63-66465

Non-Patent Documents

-   Non-Patent Document 1: “Byori to Rinsyo (Pathology and Clinical    Medicine), Vol. 25, 2007, Extra Supplement, Immunohistochemistry    useful for diagnosis”, Bunkodo, 2007

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

To such a situation, with the spread of molecular target drug therapybased mainly on antibody drugs, the necessity of exact diagnosis hasbeen increasing recently for more efficient use of the molecular targetdrugs. It is required to quantitatively detect a significantly smallamount of a biomarker on a tissue section so as to perform more exactdiagnosis of a disease also in pathological diagnosis. However, inconventional methods of staining pathological tissues, it is difficultto achieve stable quantitation and trace detection capabilities.

The present invention was made in view of the above problems inconventional technologies, and an object of the present invention is toquantitatively detect a small amount of a biosubstance (biomarker).

Means for Solving the Problem

In order to solve the above problems, according to a first aspect of thepresent invention, there is provided a tissue staining method including

staining a tissue with a staining reagent which includes a particleholding plural phosphors where a biosubstance-recognizing body is boundto the particle.

According to a second aspect of the present invention, there is provideda tissue evaluation method including

staining a tissue section with a staining reagent which contains aphosphor-holding particle holding plural phosphors where abiosubstance-recognizing body is bound to the particle;

counting the number of bright spots of fluorescence in the stainedtissue section; and

evaluating an expression level of a biosubstance corresponding to thebiosubstance-recognizing body in the stained tissue section on the basisof the number of the counted bright spots.

According to a third aspect of the present invention, there is provideda biosubstance detection method for specifically detecting abiosubstance in a pathological section, and the method includes

staining the pathological section with a staining reagent and detectingthe biosubstance in the stained pathological section, and

in the staining of the pathological section, a first particle holdingplural first phosphors where a first biosubstance-recognizing body isbound to the first particle and a second particle holding plural secondphosphors having a fluorescence wavelength different from a fluorescencewavelength of the first phosphor where a second biosubstance-recognizingbody is bound to the second particle are used as the staining reagent.

Effects of the Invention

According to the first aspect of the present invention, a stainingreagent containing a particle holding plural phosphors where abiosubstance-recognizing body is bound to the particle is used. Thus,brightness per particle is high in fluorescence observation, and a smallamount of the biosubstance can be quantitatively detected with highsensitivity.

According to the second aspect of the present invention, a small amountof a biosubstance can be quantitatively detected, and further, anexpression level of the biosubstance is evaluated on the basis of thenumber of counted bright spots in a tissue section. As a result, stableevaluation results can be achieved in the quantitative evaluation of thebiosubstance.

According to the third aspect of the present invention, a small amountof a biosubstance can be quantitatively detected, and further, particlesholding plural phosphors which type has a fluorescence wavelengthdifferent from each other where different biosubstance-recognizingbodies are bound to the particles are used. As a result, differentbiosubstances can be detected by a single pathological section.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 This is a graph showing changes in the number of particles andbrightness per cell with time.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments for implementing the present invention will bedescribed. However, the present invention is not limited thereto.

The embodiments provide a tissue staining method, and provide a tissueevaluation method and a biosubstance detection method, each of whichuses the tissue staining method.

In the tissue staining method according to the embodiments,phosphor-holding nanoparticles to which biosubstance-recognizing bodiesare bound are used.

In the tissue evaluation method according to the embodiments, phosphorsor phosphor-holding particles to which biosubstance-recognizing bodiesare bound is used, and the number of the phosphors or thephosphor-holding particles which are bound to the biomarker present onthe tissue section is determined on the basis of the number of brightspots.

The biosubstance detection method according to the embodimentsspecifically detects a biosubstance in a pathological section andbasically includes a step (1) of staining a pathological section with astaining reagent and a step (2) of detecting the biosubstance in thestained pathological section.

In particular, in the step (2), two types of nanoparticles are used asthe staining reagents.

In one type of the nanoparticles, certain biosubstance-recognizingbodies are bound to the nanoparticles, and the nanoparticles containcertain phosphors. In the other type of the nanoparticles,biosubstance-recognizing bodies different from thebiosubstance-recognizing bodies of the above type of the nanoparticlesare bound to the nanoparticles, and the nanoparticles contain phosphorshaving a fluorescence wavelength different from a fluorescencewavelength of the phosphors of the above type of the nanoparticles. Thatis, each type of biosubstance-recognizing bodies different from eachother are bound to each type of the nanoparticles, and each type of thenanoparticles contain phosphors having fluorescence wavelengthsdifferent from each other. Consequently, two different biosubstancescorresponding to the biosubstance-recognizing bodies can be detected onthe basis of the difference in fluorescence wavelengths of thephosphors. Furthermore, an antigen that has not been identified yet maybe specified in the future by selecting biosubstance-recognizing bodies.

In a preferred embodiment of the present invention, use of two types ofnanoparticles is exemplified. Alternatively, three or more types ofnanoparticles may be used to detect three or more biosubstances as longas their biosubstance-recognizing bodies are different from one anotherand phosphors (fluorescence wavelengths) are different from each other.

The details of types and characteristics of the phosphors and the likeand the biosubstance detection method are as follows.

[Phosphor]

As for the phosphors used in the present invention, organic fluorescentdyes, quantum dots (semiconductor particles), and particles ofrare-earth elements can be given as examples. The phosphors preferablyemit visual to near-infrared light having a wavelength in the range of400 to 900 nm when excited with ultraviolet to near-infrared lighthaving a wavelength in the range of 200 to 700 nm.

As for the organic fluorescent dye, fluorescein dye molecules, rhodaminedye molecules, Alexa Fluor (manufactured by Invitrogen Corporation) dyemolecules, BODIFY (manufactured by Invitrogen Corporation) dyemolecules, cascade dye molecules, coumarin dye molecules, eosin dyemolecules, NED dye molecules, pyrene dye molecules, Texas Red dyemolecules, cyanine dye molecules, and the like can be given as theexample.

Specific examples of the dye can be 5-carboxy-fluorescein,6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein,6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein,6-carboxy-2′,4,7,7′-tetrachlorofluorescein,6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, naphthofluorescein,5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodaminerhodamine 6G, tetramethylrhodamine, X-rhodamine; Alexa Fluor 350, AlexaFluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, AlexaFluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, AlexaFluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, AlexaFluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, and BODIPY 650/665 (these are manufactured byInvitrogen Corporation); methoxycoumarin, eosin, NED, pyrene, Cy5,Cy5.5, Cy7, and the like. These dyes may be used alone or in combinationof two or more.

As for the quantum dot, either of quantum dots containing a II-VI groupcompound, a III-V group compound, or a IV group element as a component(also respectively referred to as “II-VI group quantum dot”, “III-Vgroup quantum dot”, and “IV group quantum dot”) can be used. Thesequantum dots may be used alone or in combination of two or more.

Specific examples of the quantum dots can be, but not limited to, CdSe,CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, andGe.

A quantum dot constituted by the above-mentioned quantum dot as a coreand a shell covering the core can also be used. Hereinafter, throughoutthe description, the quantum dot having a shell is represented by, forexample, CdSe/ZnS for a combination of a CdSe core and a ZnS shell. Forexample, CdSe/ZnS, CdS/ZnS, InF/ZnS, InGaP/ZnS, Si/SiO₂, Si/ZnS,Ge/GeO₂, or Ge/ZnS can be used, but the quantum dot is not limitedthereto,

The quantum dot whose surface is treated with an organic polymer or thelike may be used, if necessary. For example, CdSe/ZnS having carboxygroups on the surface (manufactured by Invitrogen Corporation) andCdSe/ZnS having amino groups on the surface (manufactured by InvitrogenCorporation) can be given as examples.

As for the rare-earth element, neodymium oxide, neodymium chloride,neodymium nitrate, ytterbium oxide, ytterbium chloride, ytterbiumnitrate, lanthanum oxide, lanthanum chloride, lanthanum nitrate, yttriumoxide, yttrium chloride, yttrium nitrate, praseodymium chloride, erbiumchloride, orthophosphoric acid, ammonium phosphate, ammonium dihydrogenphosphate, or the like can be used.

[Particle which Holds Plural Phosphors]

In the present invention, a particle which holds plural phosphors refersto a nanoparticle which disperses phosphors therein (nanoparticle whichcontains plural phosphors therein (phosphor-containing nanoparticle)), aparticle which holds phosphors thereon, or a particle which holdsphosphors therein and thereon. The nanoparticle may be chemically boundto the phosphors itself or not.

The nanoparticle may be composed of any material without particularlimitation, and such as polystyrene, polylactic acid, and silica can begiven as examples.

The particles which holds plural phosphors used in the present inventioncan be prepared by any known method.

For example, organic fluorescent dye-containing silica nanoparticles canbe synthesized with reference to the synthesis of FITC-containing silicaparticles described in Langmuir, vol. 8, p. 2921, (1992). Variousorganic fluorescent dye-containing silica particles can be synthesizedwith a desired organic fluorescent dye instead of FITC.

Quantum dot-containing silica nanoparticles can be synthesized withreference to the synthesis of CdTe-containing silica nanoparticlesdescribed in New Journal of Chemistry, vol. 33, p. 561, (2009).

Silica nanoparticles which hold quantum dots thereon can be synthesizedwith reference to the synthesis of silica nanoparticles containingparticles of CdSe/ZnS capped with 5-amino-1-pentanol and APS on thesurfaces thereof described in Chemical Communication, p. 2670, (2009).

Organic fluorescent dye-containing polystyrene nanoparticles can beprepared by copolymerization of an organic dye having polymerizablefunctional groups described in U.S. Pat. No. 4,326,008 (1982) orimpregnation of polystyrene nanoparticles with an organic fluorescentdye described in U.S. Pat. No. 5,326,692 (1992).

Quantum dot-containing polymer nanoparticles can be prepared byimpregnation of polystyrene nanoparticles with quantum dots described inNature Biotechnology, vol. 19, p. 631, (2001).

The particles which hold plural phosphors used in the present inventionmay have any average particle diameter without particular limitation,for example, an average particle diameter of about 30 to 800 nm. If theaverage particle diameter is less than 30 nm, the amount of phosphors ofthe particles is insufficient for quantitative evaluation of a targetbiosubstance. If the average particle diameter exceeds 800 nm, theparticles cannot be readily bound to a biosubstance in a pathologicaltissue. The average particle diameter is preferably in the range of 40to 500 nm. The reason why the average particle diameter is determined asfrom 40 to 500 nm is that an average particle diameter less than 40 nmrequires an expensive detection system, while an average particlediameter excess 500 nm narrows a range of the quantitative determinationdue to its physical size. The particles may have any coefficient ofvariation (=(standard deviation/average value)×100%), which shows adistribution in particle diameter, without particular limitation.Particles having a coefficient of variation of 20% can also be used. Theaverage particle diameter is determined by taking an electron micrographwith a scanning electron microscope (SEM), measuring cross-sectionalareas of a sufficient number of particles, and using the diameters ofcircles each having the same area as the measured cross-sectional area.In the present application, the arithmetic average of particle diametersof 1000 particles is defined as the average particle diameter. Thecoefficient of variation is calculated also from the particle sizedistribution of 1000 particles.

[Buffer Solution]

A buffer solution is a solvent for stably maintaining a environmentsuitable for an antigen-antibody reaction. For example, phosphate bufferphysiological saline solutions (PBSs), phosphate buffer solutions, Trisbuffer solutions, MES buffer solutions, and citrate-phosphate buffersolutions, and the like can be given as examples.

[Binding of Biosubstance-Recognizing Bodies to Phosphor-HoldingNanoparticles]

The biosubstance-recognizing body according to the present invention isa body which is specifically bound to and/or reacts with a targetbiosubstance. For example, nucleotide chains, proteins, antibodies, andthe like can be given as examples. Specific examples of the body can bean anti-HER2 antibody which is specifically bound to a protein, HER2,present on cell surfaces; an anti-ER antibody which is specificallybound to an estrogen receptor (ER) present on cell nuclei; anti-actinantibody which is specifically bound to actin forming cytoskeletons; andthe like. In particular, phosphor-holding nanoparticles to which theanti-HER2 antibodies and the anti-ER antibodies are bound are suitable,because such nanoparticle can be used for selection of a drug for breastcancer.

Biosubstance-recognizing bodies may be bound to a phosphor-holdingnanoparticle in any form without particular limitation, and such ascovalent bonding, ion bonding, hydrogen bonding, coordinate bonding,physisorption, and chemisorption can be given as examples. In light ofthe stability of binding, bonding having a strong binding force such ascovalent bonding is suitable.

Furthermore, any organic molecule for linking biosubstance-recognizingbodies and a phosphor-holding nanoparticle may be used. For example, inorder to inhibit non-specific adsorption to a biosubstance, apolyethylene glycol chain can be used, and SM(PEG)12 manufactured byThermo Scientific Inc. can be used.

In binding of biosubstance-recognizing bodies to phosphor-holding silicananoparticles, the same procedure can be used in all cases of using anorganic fluorescent dye, quantum dots, or particles of a rare-earthelement as the phosphor.

For example, a silane coupling agent, which is a compound widely usedfor binding an inorganic material with an organic material, can be used.The silane coupling agent is a compound having an alkoxysilyl groupwhich gives a silanol group through hydrosis at one end of the moleculeand a functional group at the other end, such as a carboxyl group, anamino group, an epoxy group, or an aldehyde group. The silane couplingagent is bound to an inorganic material via the oxygen atom of thesilanol group.

Specifically, mercaptopropyltriethoxysilane,glycidoxypropyltriethoxysilane, aminopropyltriethoxysilane, silanecoupling agents having polyethylene glycol chains (e.g., PEG-silane no.SIM6492.7 manufactured by Gelest Inc.), and the like are given asexamples of the silane coupling agent. When the silane coupling agentsis used, two or more types thereof can be used in combination.

The reaction procedure of the silane coupling agent with organicfluorescent dye-holding silica nanoparticles may be performed by anyknown method.

For example, prepared organic fluorescent dye-holding silicananoparticles are dispersed in pure water, and thenaminopropyltriethoxysilane is added thereto for a reaction at roomtemperature for 12 hours. After completion of the reaction,centrifugation or filtration is performed to obtain organic fluorescentdye-holding silica nanoparticles having the surfaces modified withaminopropyl groups. Subsequently, the amino group is made to react witha carboxyl group of an antibody to bind the antibody to the organicfluorescent dye-holding silica nanoparticle via an amide bond.Furthermore, a condensing agent such as1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC,manufactured by Pierce) may be used as needed.

If necessary, a linker compound having a site that can be directly boundto an organic fluorescent dye-holding silica nanoparticle modified withan organic molecule and a site that can be bound to a target materialmolecule can be used.

Specifically, by using sulfosuccinimidyl4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC,manufactured by Pierce) having a site that selectively reacts with anamino group and a site that selectively reacts with a mercapto group,the amino group of an organic fluorescent dye-holding silicananoparticle modified with aminopropyltriethoxysilane is bound to themercapto group of an antibody to provide organic fluorescent dye-holdingsilica nanoparticles to which the antibodies are bound.

Binding of biosubstance-recognizing bodies to a phosphor-holdingpolystyrene nanoparticle can be achieved by substantially the sameprocedure in all cases of using an organic fluorescent dye, quantumdots, and particles of a rare-earth element as the phosphor. That is,polystyrene nanoparticles having functional groups such as amino groupsare impregnated with an organic fluorescent dye, quantum dots, orparticles of a rare-earth element to provide phosphor-holdingpolystyrene particles having functional groups. A subsequent use of EDCor sulfo-SMCC can provide phosphor-holding polystyrene particles towhich the antibodies are bound.

[Staining Method (Biosubstance Detection Method)]

Hereinafter, a staining method (biosubstance detection method) of thepresent invention will be described.

The staining method of the present invention can be applied to not onlypathological tissue sections but also cell staining.

The section to which the staining method of the present invention isapplied may be prepared by any known method without particularlimitation.

1) Deparaffinization Step

A pathological section is immersed in xylene in a container to removeparaffin. The immersion may be performed at any temperature withoutparticular limitation, for example, at room temperature. The immersiontime is preferably 3 minutes or more and 30 minutes or less. The xylenemay be replaced with new xylene during the immersion if necessary.

Subsequently, the pathological section is immersed in ethanol in acontainer to remove xylene. The immersion may be performed at anytemperature without particular limitation, for example, at roomtemperature. The immersion time is preferably 3 minutes or more and 30minutes or less. The ethanol may be replaced with new ethanol during theimmersion if necessary.

Subsequently, the pathological section is immersed in water in acontainer to remove ethanol. The immersion may be performed at anytemperature without particular limitation, for example, at roomtemperature. The immersion time is preferably 3 minutes or more and 30minutes or less. The water may be replaced with new water during theimmersion if necessary.

2) Activation Treatment

In accordance with a known method, a target biosubstance is subjected toactivation treatment.

The activation can be performed under any condition without limitation.As for an activating solution, a 0.01 M citrate buffer solution (pH6.0), a 1 mM EDTA solution (pH 8.0), 5% urea, a 0.1 M Tris-hydrochloridebuffer solution, or the like can be used. As for a heater, an autoclave,a microwave heater, a pressure cooker, a water bath, or the like can beused. The activation may be performed at any temperature withoutparticular limitation, for example, at room temperature. The temperaturemay range from 50 to 130° C., and the time may range from 5 to 30minutes.

Subsequently, the section after the activation treatment is washed bybeing immersed in water and PBS in containers. The washing may beperformed at any temperature without particular limitation, for example,at room temperature. Each immersion time is preferably 3 minutes or moreand 30 minutes or less. The PBS may be replaced with new PBS during theimmersion if necessary.

3) Staining with Phosphor-Holding Nanoparticles to whichBiosubstance-Recognizing Bodies are Bound

A PBS dispersion of phosphor-holding nanoparticles to whichbiosubstance-recognizing bodies are bound is placed on a pathologicalsection to react with the target biosubstance. Various biosubstances canbe stained by changing the biosubstance-recognizing bodies bound to thephosphor-holding nanoparticles.

In order to simultaneously detect (two or more) different biosubstances,PBS dispersions of the phosphor-containing nanoparticles to whichdifferent biosubstance-recognizing bodies are bound are individuallyprepared, and the dispersions are placed on a pathological section toreact with target biosubstances. In the placement on the pathologicalsection, the PBS dispersions of the respective phosphor-containingnanoparticles may be mixed before being placed on a pathological sectionor may be separately placed on a pathological section. The dispersionsmay be mixed at any mixing ratio without particular limitation, and theratio may range from 1:1 to 5:1 for achieving the advantageous effect ofthe present invention.

The PBS dispersion of the phosphor-holding nanoparticles may contain aknown blocking agent such as BSA-containing PBS and a surfactant such asTween 20.

The staining may be performed at any temperature without particularlimitation, for example, at room temperature. The reaction time ispreferably 30 minutes or more and 24 hours or less.

It is preferred to apply dropwise a known blocking agent such asBSA-containing PBS to the section before the staining with thephosphor-holding nanoparticles.

Subsequently, the stained section is immersed in PBS in a container toremove unreacted phosphor-holding nanoparticles. The PBS solution maycontain a surfactant such as Tween 20. The removal may be performed atany temperature without particular limitation, for example, at roomtemperature. The immersion time is preferably 3 minutes or more and 30minutes or less. The PBS may be replaced with new PBS during theimmersion if necessary.

In order to observe the tissue morphology, hemotoxylin-eosin stainingmay be performed.

A cover glass is placed on the section for sealing. A commerciallyavailable sealing agent may be used as needed.

4) Observation with Fluorescence Microscope

The stained pathological section observed with a fluorescencemicroscope, and an expression level of the target biosubstance isevaluated on the basis of the number of bright spots or luminescentbrightness.

In the count of the number of bright spots or the measurement ofluminescent brightness, an excitation light source and a fluorescencedetection optical filter are selected so as to correspond to anabsorption maximum wavelength and a fluorescence wavelength of usedphosphors.

The number of bright spots or luminescent brightness can be counted ormeasured using an image-analyzing software, e.g., free analysis softwareImageJ, or software G-count manufactured by G-Angstrom, forautomatically counting all bright spots.

Examples in which the present invention is specifically implemented onthe basis of the above-described embodiments will be described, but thepresent invention should not be limited thereto.

Example 1 Procedure 1: Synthesis of Phosphor-Containing Particles

An organoalkoxysilane compound was obtained by mixing 6.6 mg oftetramethylrhodamine (TAMRA-SE, manufactured by Invitrogen Corporation)and 3 μL of 3-aminopropyltrimethoxysilane (KBM903, manufactured byShin-Etsu Silicone) in DMF, Then, 0.6 mL of the obtainedorganoalkoxysilane compound was mixed with 48 mL of ethanol, 0.6 mL oftetraethoxysilane (TEOS), 2 mL of water, and 2 mL of 28% aqueous ammoniafor 3 hours.

The liquid mixture prepared in the above step was centrifuged at 10000 Gfor 20 minutes, and the supernatant was removed. Thereafter, ethanol wasadded to disperse the precipitate therein, and the dispersion wascentrifuged again. The precipitate was washed twice with each of ethanoland pure water by the same procedure.

The obtained tetramethylrhodamine-containing silica nanoparticles wereobserved with an SEM to measure particle diameters of 200 particles. Theaverage particle diameter was 104 nm, and the coefficient of variationwas 12%.

Cy5-containing silica nanoparticles having average particle diameters of20, 42, 103, 204, and 498 nm were prepared using Cy5-SE (manufactured byRoche) through the same method.

Furthermore, FITC-containing silica nanoparticles having an averageparticle diameter of 106 nm were prepared using FITC-SE (manufactured byInvitrogen Corporation) through the same method.

Procedure 2: Binding of Antibodies to Phosphor-Containing Particles

Each concentration of the phosphor-containing silica nanoparticlesprepared in Procedure 1 (the tetramethylrhodamine-containing silicananoparticles, the Cy5-containing silica nanoparticles, and theFITC-containing silica nanoparticles) was adjusted to 3 nM with aphosphate buffer physiological saline solution (PBS) containing 2 mMethylenediaminetetraacetic acid (EDTA). This solution was mixed with afinal concentration of 10 mM of SM(PEG)12(succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester,manufactured by Thermo Scientific Inc. Inc.), followed by reaction for 1hour. This solution mixture was centrifuged at 10000 G for 20 minutes,and the supernatant was removed. Thereafter, PBS containing 2 mM EDTA isadded to disperse the precipitate therein. The dispersion wascentrifuged again. The precipitate was washed three times by the sameprocedure to obtain silica nanoparticles for antibody binding.

Meanwhile, anti-human ER antibodies were subjected to reductiontreatment with 1 M dithiothreitol (DTT), and excess DTT was removed by agel filtration column to obtain a solution of the reduced antibodiescapable of bonding to the silica particles.

The silica nanoparticles for antibody binding and the reduced antibodysolution obtained above were mixed with each other in PBS containing 2mM EDTA, followed by reaction for 1 hour. The reaction was stopped byadding 10 mM mercaptoethanol. Then, the obtained solution wascentrifuged at 10000 G for 20 minutes, and the supernatant was removed.Thereafter, PBS containing 2 mM EDTA was added to disperse theprecipitate therein. The dispersion was centrifuged again. Theprecipitate was washed three times by the same procedure to obtainphosphor-containing silica nanoparticles to which the anti-human ERantibodies were bound.

Procedure 3: Tissue Staining with Phosphor-Containing Particles

Human mammary tissue was immunostained with the phosphor-containingsilica nanoparticles to which the anti-human ER antibodies prepared inProcedure 2 were bound. A tissue array slide (CB-A712) manufactured byCosmo Bio Co., Ltd. was used as a section to be stained. The tissuearray slide was deparaffinized, and washed by being immersed in xylene,ethanol, and water which were substituted in this order, andsubsequently subjected to autoclave treatment in a 10 mM citrate buffersolution (pH 6.0) for 15 minutes to perform the activation treatment ofthe antigen. The tissue array slide after the activation treatment ofthe antigen was washed with a PBS buffer solution and was subjected toblocking treatment with a PBS buffer solution containing 1% BSA in amoist chamber for 1 hour.

After the blocking treatment, each type of the phosphor-containingsilica nanoparticles to which the anti-human ER antibodies were boundwas diluted with a PBS buffer solution containing 1% BSA to 0.05 nM andwas made to with the tissue section for 3 hours. After the reaction withthe phosphor-containing silica nanoparticles to which the anti-human ERantibodies were bound, the tissue array slide was washed with a PBSbuffer solution and was sealed with Aquatex manufactured by MerckChemicals.

Procedure 4: Count of Bright Spots in Tissue Stained withPhosphor-Containing Particles

An image of the tissue section stained in Procedure 3 was taken using aDSU confocal microscope manufactured by Olympus Corporation, and brightspots were counted with a bright spot-counting software G-countmanufactured by G-Angstrom.

Cy5 was observed with a filter set of an excitation filter (640/30 nmband path filter), a beam splitter (660 nm), and a fluorescence filter(690/50 nm band path filter).

Tetramethylrhodamine was observed with a filter set of an excitationfilter (550/25 nm band path filter), a beam splitter (570 nm), and afluorescence filter (605/70 nm band path filter).

FITC was observed with a filter set of an excitation filter (470/40 nmband path filter), a beam splitter (495 nm), and a fluorescence filter(525/50 nm band path filter).

The number of bright spots in 30 cells was counted for each of eightareas in the tissue array slide which areas were anticipated to showdifferent staining concentrations in preliminary DAB staining todetermine the number (mean value) of bright spots per cell. Similarly,luminescent brightness of 30 cells was measured for each of the eightareas to determine luminescent brightness (mean value) per cell.

Comparative Example Tissue Staining with Fluorescent Dye Alone

As a comparative example, a tissue array slide was stained as inProcedure 3 with anti-human ER antibodies bound to fluorescent dye aloneof Cy5, tetramethylrhodamine, or FITC. Bright spots in the tissue werecounted as in Procedure 4.

Specifically, eight areas in the tissue array slide were each measuredfor the number of bright spots and luminescent brightness of 30 cellsfor each dye to determine the number of bright spots per cell andluminescent brightness per cell.

[Experimental Results A]

A difference in detection sensitivity for the biomarker (ER) due to adifference in fluorescent dye contained in the labeling object (i.e.,difference in emission wavelength) was examined.

Table 1 shows the numbers of bright spots per cell counted in the casesof using Cy5-containing silica nanoparticles (average particle diameter:103 nm), tetramethylrhodamine-containing silica nanoparticles (averageparticle diameter: 104 nm) and FITC-containing silica nanoparticles(average particle diameter: 106 nm). In Table 1, “−” denotes that nobright spot having a higher brightness than the background level waspresent, and “+” denotes that the light emission was too strong to bedistinguished from bright spots on the periphery.

TABLE 1 LUMINESCENCE WAVELENGTH OF LABELING OBJECT AND THE NUMBER OFBRIGHT SPOTS PER CELL AREA NUMBER LABELING OBJECT 1 2 3 4 5 6 7 8Cy5-containing silica − 4 14 32 120 392 + + nanoparticleTetramethylrhodamine- − 6 12 34 112 380 + + containing silicananoparticle FITC-containing silica − − 10 36 118 381 + + nanoparticle

As shown in Table 1, in all the phosphor-containing particles, thebiomarker can be quantitatively evaluated on the basis of the differenceof the number of the bright spots. In the tissue section of area No. 2,however, bright spots higher than the background level were not countedin the case of using the FITC-containing nanoparticles, whereas thenumber of bright spots were counted in the cases of using theCy5-containing silica nanoparticles and thetetramethylrhodamine-containing silica nanoparticles. Thus, it isrevealed that the particles containing a fluorescent dye having a longerexcitation wavelength, Cy5 (excitation wavelength: 650 nm, emissionwavelength: 670 nm) or tetramethylrhodamine (excitation wavelength: 550nm, emission wavelength: 570 nm) can detect a smaller amount ofbiomarker compared with the particles containing FITC (excitationwavelength: 495 nm, emission wavelength: 520 nm).

[Experimental Results B]

A difference in detection sensitivity for the biomarker (ER) due to adifference in particle diameter of the labeling object was examined.

Table 2 shows the numbers of the bright spots per cell counted in thecases of using each types of the Cy5-containing silica nanoparticles(average particle diameter: 20, 42, 103, 204, and 498 nm) and Cy5 dyealone (Comparative Example). In Table 2, “−” denotes that no bright spothaving a higher brightness than the background level was present, and“+” denotes that the light emission was too strong to be distinguishedfrom bright spots on the periphery.

TABLE 2 PARTICLE DIAMETER OF LABELING OBJECT AND THE NUMBER OF BRIGHTSPOTS PER CELL AREA NUMBER LABELING OBJECT 1 2 3 4 5 6 7 8 Cy5 dye alone− − − − + + + + Cy5-containing silica − − − − + + + +nanoparticle/average particle diameter: 20 nm Cy5-containing silica − −12 39 129 406 + + nanoparticle/average particle diameter: 42 nmCy5-containing silica − 4 14 32 120 392 + + nanoparticle/averageparticle diameter: 103 nm Cy5-containing silica − 4 10 36 114 383 + +nanoparticle/average particle diameter: 204 nm Cy5-containing silica − 613 33 122 + + + nanoparticle/average particle diameter: 498 nm −: nobright spot having a higher brightness than the background level waspresent +: the light emission was too strong to be distinguished frombright spots on the periphery

As shown in Table 2, in the cases of using the Cy5-containing silicananoparticles having an average particle diameter of 42, 103, 204, or498 nm, it was possible to quantitatively evaluate the biomarker on thebasis of the difference in the number of the bright spots in each areawhere it was possible to count the number of the bright spots. However,in the case of using the Cy5-containing silica nanoparticles having anaverage particle diameter of 498 nm, it was not possible to distinguishbright spots from one another in the tissue section of area No. 6. Thus,it is revealed that the quantification range is narrower when abiomarker is expressed with a high frequency.

in addition, in the cases of using the Cy5 dye alone and theCy5-containing silica nanoparticles having an average particle diameterof 20 nm, no bright spot higher than background level was present in thetissue sections of area Nos. 1 to 4, and bright spots were notdistinguished from those on the periphery in the tissue sections of areaNos. 5 to 8. Thus, it is revealed that a small amount of biomarkercannot be quantitatively evaluated by a level of bright spots.

[Experimental Results C]

Detection sensitivities of fluorescent dye-containing particles andfluorescent dye alone for a biomarker (ER) were compared by the numberof bright spots.

Table 3 shows the numbers of the bright spots per cell that were countedin each case of using Cy5-containing silica nanoparticles (averageparticle diameter: 103 nm), tetramethylrhodamine-containing silicananoparticles (average particle diameter 104 nm), FITC-containing silicananoparticles (average particle diameter: 106 nm) Cy5,tetramethylrhodamine, or FITC. In Table 3, “−” denotes that no brightspot having a higher brightness than the background level was present,and “+” denotes that the light emission was too strong to bedistinguished from bright spots on the periphery.

TABLE 3 COMPARISON OF STAINING BETWEEN DYE- CONTAINING PARTICLE AND DYEALONE (THE NUMBER OF BRIGHT SPOTS) AREA NUMBER LABELING OBJECT 1 2 3 4 56 7 8 Cy5-containing silica − 4 14 32 120 392 + + nanoparticleTetramethylrhodamine- − 6 12 34 112 380 + + containing silicananoparticle FITC-containing silica − − 10 36 118 381 + + nanoparticleCy5 − − − − + + + + Tetramethylrhodamine − − − − + + + + FITC − − −− + + + + −: no bright spot having a higher brightness than thebackground level was present +: the light emission was too strong to bedistinguished from bright spots on the periphery

As shown in Table 3, in the ease of tissue staining using fluorescentdye alone, no bright spot in the tissue sections of area Nos. 1 to 4having a brightness higher than the background level was present, andbright spots in the tissue sections of area Nos. 5 to 8 were notdistinguished from those on the periphery. Thus, it is revealed that asmall amount of biomarker cannot be quantitatively evaluated by a levelof bright spots.

In contrast, in the cases of using phosphor-containing particles as thelabeling object, a small amount of biomarker can also be quantitativelydetermined with high accuracy.

[Experimental Results D]

Detection sensitivities of fluorescent dye-containing particles andfluorescent dye alone for a biomarker (ER) were compared by luminescentbrightness.

Table 4 shows the luminescent brightness per cell measured on the basisof image data obtained using a DSU confocal microscope in each case ofusing Cy5-containing nanoparticles (average particle diameter: 103 nm),tetramethylrhodamine-containing silica nanoparticles (average particlediameter: 104 nm) FITC-containing silica nanoparticles (average particlediameter: 106 nm) Cy5, tetramethylrhodamine, or FITC. The unit ofluminescent brightness is a.u. (arbitrary unit). In Table 4, “0” denotesthat the light emission was lower than the background level.

TABLE 4 COMPARISON OF STAINING BETWEEN DYE-CONTAINING PARTICLE AND DYEALONE (LUMINESCENT BRIGHTNESS) AREA NUMBER LABELING OBJECT 1 2 3 4 5 6 78 Cy5-containing silica 0 42 160 390 1280 3920 9860 12333 nanoparticleTetramethylrhodamine- 0 60 143 366 1190 3686 10024 11369 containingsilica nanoparticle FITC-containing silica 0 0 112 382 1220 3842 1021112488 nanoparticle Cy5 0 0 0 0 38 118 260 360 Tetramethylrhodamine 0 0 00 33 129 248 393 FITC 0 0 0 0 24 120 263 386 0: the light emission waslower than the background level

As shown in Table 4, it is revealed that a smaller amount of a biomarkercan be detected by using phosphor-containing particles compared to thecase of using fluorescent dye alone.

As described above, the use of particles holding plural phosphors wherea biosubstance-recognizing bodies are bound to the particles as astaining reagent increases brightness per particle in fluorescenceobservation of a tissue section, and thereby allows quantitativedetection of a small amount of a biomarker (biosubstance correspondingto the biosubstance-recognizing body) with high sensitivity.

In addition, in the use of particles containing plural phosphors, sincethe phosphors are present inside the particle, the durability of thephosphors is enhanced.

Example 2 Procedure 1: Synthesis of Phosphor-Holding Particles

An organoalkoxysilane compound was obtained by mixing 6.6 mg oftetramethylrhodamine (TAMRA-SE, manufactured by Invitrogen Corporation)(excitation wavelength: 550 nm, emission wavelength: 570 nm) and 3 μL of3-aminopropyltrimethoxysilane (KBM903, manufactured by Shin-EtsuSilicone) in DMF. Then, 0.6 mL of the obtained organoalkoxysilanecompound was mixed with 48 mL of ethanol, 0.6 mL of tetraethoxysilane(TEOS), 2 mL, of water, and 2 mL of 28% aqueous ammonia for 3 hours.

The liquid mixture prepared by the above steps was centrifuged at 10000G for 20 minutes, and the supernatant was removed. Thereafter, ethanolwas added to disperse the precipitate therein, and the liquid mixturewas centrifuged again. The precipitate was washed twice with each ofethanol and pure water by the same procedure.

The obtained tetramethylrhodamine-holding silica nanoparticles wereobserved with an SEM to measure particle diameters of 200 particles. Theaverage particle diameter was 104 nm, and the coefficient of variationwas 12%.

Cy5-holding silica nanoparticles having an average particle diameter of103 nm were obtained using Cy5-SE (manufactured by Roche) (excitationwavelength: 650 nm, emission wavelength: 670 nm) by the same method.

Procedure 2: Binding of Antibodies to Phosphor-Holding Particle andQuantum Dot

Each concentration of the phosphor-holding silica nanoparticles preparedin Procedure 1 (the tetramethylrhodamine-holding silica nanoparticlesand the Cy5-holding nanoparticles) was adjusted to 3 nM with a phosphatebuffer physiological saline solution (PBS) containing 2 mMethylenediaminetetraacetic acid (EDTA). This solution was mixed with afinal concentration of 10 mM of SM(PEG)12(succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester,manufactured by Thermo Scientific Inc. Inc.), followed by reaction for 1hour. This solution mixture was centrifuged at 10000 G for 20 minutes,and the supernatant was removed. Thereafter, PBS containing 2 mM EDTAwas added to disperse the precipitate therein. The dispersion wascentrifuged again. The precipitate was washed three times by the sameprocedure to obtain silica nanoparticles for antibody binding.

Meanwhile, anti-human ER antibodies were subjected to reductiontreatment with 1 M dithiothreitol (DTT), and excess DTT was removed by agel filtration column to obtain a solution of reduced antibodies capableof bonding to silica particles.

The silica nanoparticles for antibody binding and the reduced antibodysolution obtained above were mixed with each other in PBS containing 2mM EDTA, followed by reaction for 1 hour. The reaction was stopped byadding 10 mM mercaptoethanol. Thereafter, the obtained solution wascentrifuged at 10000 G for 20 minutes, and the supernatant was removed.Thereafter, PBS containing 2 mM EDTA was added to disperse theprecipitate therein. The solution was centrifuged again. The precipitatewas washed three times by the same procedure to obtain phosphor-holdingsilica nanoparticles to which the anti-human ER antibodies were bound.

Anti-human. ER antibodies were bound to quantum dots using a QD655antibody labeling kit (Q22021MP) manufactured by Invitrogen Corporation.The binding of the antibodies was performed as prescribed in the kitthrough a step of activating the quantum dots by SMCC and a step ofreducing the antibodies by DTT.

Procedure 3: Tissue Staining with Phosphor-Holding Particles or QuantumDots

Human mammary tissue was immunostained with the phosphor-holding silicananoparticles to which the anti-human ER antibodies were bound, and withthe quantum dots to which the anti-human ER antibodies prepared inProcedure 2 were bound. A tissue array slide (CB-A712) manufactured byCosmo Bio Co., Ltd was used as a section to be stained. The tissue arrayslide was deparaffinized, was then washed by being immersed in xylene,ethanol, and water which were substituted in this order, and wassubjected to autoclave treatment in a 10 mM citrate buffer solution (pH6.0) for 15 minutes to activate the antigen. The tissue array slideafter the activation treatment of the antigen was washed with a PBSbuffer solution and was subjected to blocking treatment with a PBSbuffer solution containing 1% BSA in a moist chamber for 1 hour.

After the blocking treatment, the phosphor-holding silica nanoparticlesto which the anti-human ER antibodies were bound and the quantum dots towhich the anti-human ER antibodies were bound were each diluted with aPBS buffer solution containing 1% BSA to 0.05 nM and were made to reactwith the tissue section for 3 hours. Subsequently, the tissue arrayslide was washed with a PBS buffer solution and was sealed with Aquatexmanufactured by Merck Chemicals.

Procedure 4: Count or Measurement of Bright Spots, Brightness, and theNumber of Particles in Stained Tissue

An image of the tissue section stained in Procedure 3 was taken using aDSU confocal microscope manufactured by Olympus Corporation, and brightspots were counted with a bright spot-counting software G-countmanufactured by G-Angstrom.

Cy5 was observed with a filter set of an excitation Filter (640/30 nmband path filter), a beam splitter (660 nm), and a fluorescence filter(690/50 nm band path filter).

Tetramethylrhodamine was observed with a filter set of an excitationfilter (550/25 nm band path filter), a beam splitter (570 nm), and afluorescence filter (605/70 nm band path filter).

QD655 was observed with a filter set of an excitation filter (350/50 nmband path filter), a beam splitter (400 nm) and a fluorescence filter(590 nm long path filter).

For each of eight areas in the tissue array slide which areas wereanticipated to show different staining concentrations in preliminary DABstaining, the number of bright spots in 60 cells was counted andbrightness of each bright spot was measured.

[Experimental Results A]

First, brightness per particle was determined on the basis of brightnessdistribution of bright spots (the number of bright spots at eachbrightness level) obtained in each case of using the phosphor-holdingparticles or the quantum dots. Specifically, the brightness of thehighest frequency in the brightness distribution was determined as thebrightness per particle.

The bright, spots in each of eight areas (60 cells in each area) werecounted in each case of using the Cy5-holding silica nanoparticles, thetetramethylrhodamine-holding silica nanoparticles, or QD655. As aresult, the number of the bright spots having a brightness of 82 was thelargest in the measurement using the Cy5-holding silica nanoparticles;the number of the bright spots having a brightness of 69 was the largestin the measurement using the tetramethylrhodamine-holding silicananoparticles; and the number of the bright spots having a brightness of64 was the largest in the measurement using QD655.

Table 5 shows the brightness distribution of all bright spots in theeight areas (60 cells×8 areas) in each case of using the Cy5-holdingsilica nanoparticles, the tetramethylrhodamine-holding silicananoparticles, or QD655. The unit of brightness is a.u. (arbitraryunit). Table 5 shows the number of the bright spots in each brightnessrange of 0 to 30, 31 to 60, 61 to 90, 91 to 120, 121 to 150, 151 to 180,181 to 210, or 211 to 255.

TABLE 5 DISTRIBUTION OF BRIGHTNESS PER BRIGHT SPOT BRIGHTNESS LABELING0- 31- 61- 91- 121- 151- 181- 211- OBJECT 30 60 90 120 150 180 210 255Cy5-holding silica 4 88 814 41 62 210 42 143 nanoparticle Tetramethyl- 849 923 69 242 42 118 63 rhodamine- holding silica nanoparticle QD655 320 1120 36 242 32 96 66

As shown in Table 5, the Cy5-holding silica nanoparticles had peaks inthe brightness ranges of 61 to 90, 151 to 180, and 211 to 255. In theCy5-holding silica nanoparticles, the number of the bright spots havinga brightness of 82 was the largest. It was therefore revealed that thebrightness of the bright spots included in the brightness range of 151to 180 was the sum of two particles, and that the brightness of thebright spots included in the brightness range of 211 to 255 was the sumof three particles. Thus, the brightness of one phosphor-holdingparticle or one quantum dot can be determined on the basis of brightnessdistribution.

[Experimental Results B]

Next, the “number of particles per cell” was determined in each area bydividing the “sum of brightness per cell” in each area by the“brightness of one particle” under the assumption that brightness of oneCy5-holding silica nanoparticle was 82, brightness of onetetramethylrhodamine-holding silica nanoparticle was 69, and brightnessof one particle of QD655 was 64 on the basis of the results inExperimental results A.

Table 6 shows the numbers of the particles per cell in each area in eachcase of using the Cy5-holding silica nanoparticles, thetetramethylrhodamine-holding silica nanoparticles, or QD655. In Table 6,“−” denotes that no bright spot having a higher brightness than thebackground level was present.

TABLE 6 LUMINESCENT WAVELENGTH OF LABELING OBJECT AND THE NUMBER OFPARTICLES PER CELL AREA NUMBER LABELING OBJECT 1 2 3 4 5 6 7 8Cy5-holding silica − 4 14 32 120 392 688 811 nanoparticleTetramethylrhodamine- − 6 12 34 112 380 648 863 holding silicananoparticle QD655 − − − 22 98 421 699 920 −: no bright spot having ahigher brightness than the background level was present

As shown in Table 6, the expression level of a biomarker can bequantitatively evaluated by the difference in the number of theparticles per cell in each case of the Cy5-holding silica nanoparticles,the tetramethylrhodamine-holding silica nanoparticles, or QD655.However, in the quantitative evaluation of a further smaller amount ofbiomarker by a level of bright spots (area Nos. 2 and 3), theCy5-holding silica nanoparticles and the tetramethylrhodamine-holdingsilica nanoparticles have higher detection sensitivity than QD655.

[Experimental Results C]

Time-dependent changes in bright spots in tissue stained with thephosphor-holding particles or the quantum dots were examined.

The number of bright spots and brightness of each bright spot in 60cells in the tissue section of area No. 6 in the tissue array slide werecounted and measured in accordance with Procedure 4 on the 0, 3rd, 30th,and 90th days after preparation of the tissue section stained inaccordance with Procedure 3. Then, the number of the particles per cellwas calculated by the same way as in Experimental results B. Brightnessper cell was calculated by dividing the sum of brightness of brightspots by the number of the cells.

FIG. 1 shows time-dependent changes in the number of the particles percell and the brightness per cell calculated as for the tissue section ofarea No. 6 in each case of using the Cy5-holding silica nanoparticles,the tetramethylrhodamine-holding silica nanoparticles, or QD655.

FIG. 1 shows that the brightness per cell gradually decreased with theelapsed time from the preparation of the tissue section, whereas thenumber of the particles per cell was stable even on the 90th day afterthe preparation of the tissue section. That is, the brightness offluorescence emitted by each particle gradually decreased, whereas thenumber of the particles binding to the biomarker present in a tissuesection did not change. Consequently, quantitative evaluation of theexpression level of the biomarker on the basis of the difference in thenumber of particles per cell gives more stable results of evaluation.

[Experimental Results D]

Next, the results in the case of staining with the phosphor-holdingparticles or the quantum dots were compared with the results in the caseof using fluorescent dye alone.

As examples of using fluorescent dye alone, Cy5 to which anti-human ERantibodies were bound and tetramethylrhodamine to which anti-human ERantibodies were bound were used, and a tissue array slide was stainedtherewith by the same way as Procedure 3. The number of the bright spotsand the brightness of each bright spot in 60 cells were determined bythe same way as Procedure 4 for eight areas in the tissue array slide.The number of the particles per cell was calculated by the same way asin Experimental results B.

Table 7 shows the numbers of the particles per cell in each area in eachcase of using the Cy5-holding silica nanoparticles, thetetramethylrhodamine-holding silica nanoparticles, QD655, Cy5, ortetramethylrhodamine. In Table 7, “−” denotes that the light emissionwas lower than the background level, and “+” denotes that, although thelight emission higher than background level was present, no bright spotwas determined.

TABLE 7 COMPARISON OF STAINING BETWEEN DYE- CONTAINING PARTICLE AND DYEALONE (THE NUMBER OF PARTICLES PER CELL) AREA NUMBER LABELING OBJECT 1 23 4 5 6 7 8 Cy5-holding silica − 4 14 32 120 392 688 811 nanoparticleTetramethylrhodamine- − 6 12 34 112 380 648 863 holding silicananoparticle QD655 − − − 22 98 421 699 920 Cy5 − − − − + + + +Tetramethylrhodamine − − − − + + + + −: the light emission was lowerthan the background level +: the light emission higher than backgroundlevel was present (determination of bright spots was impossible)

Table 7 shows that the tissue staining with fluorescent dye alone cannotevaluate a biomarker quantitatively by a level of bright spots.

As described above, because an expression level of biosubstance isevaluated on the basis of the number of bright spots of fluorescencecounted for a tissue section, stable results in quantitative evaluationof a biosubstance can be obtained.

In addition, by, using phosphor-holding particles which hold pluralphosphors, brightness per particle in fluorescence observationincreases. Thus, quantitative detection of a small amount of abiosubstance with high sensitivity can be performed.

Example 3 Synthesis of Phosphor-Containing Nanoparticles a to fSynthesis Example 1 Organic Fluorescent Dye-Containing Silica: Synthesisof Cy5-Containing Nanoparticles

“Nanoparticle a” was prepared by a method of the following Steps (1) to(4):

Step (1): 1 mg (0.00126 mmol) of an N-hydroxysuccinimide esterderivative of Cy5 (manufactured by GE Healthcare) and 400 μL (1.796mmol) of tetraethoxysilane were mixed.

Step (2): 40 mL of ethanol and 10 mL of 14% aqueous ammonia were mixed.

Step (3): The solution prepared in Step (1) was added to the solutionprepared in Step (2) being stirred at room temperature. The stirring wascontinued for 12 hours from the starting of the addition.

Step (4): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, ethanol was addedto disperse the precipitate therein. The dispersion was centrifugedagain. The precipitate was washed once with each of ethanol and purewater by the same procedure.

The obtained silica nanoparticle a was observed with a scanning electronmicroscope (SEM, model S-800, manufactured by Hitachi, Ltd.). Theaverage particle diameter was 110 nm, and the coefficient of variationwas 12%.

Synthesis Example 2 Organic Fluorescent Dye-Containing Silica: Synthesisof TAMRA-Containing Silica Nanoparticles

“Nanoparticle b” was prepared by a method of the following Steps (1) to(4):

Step (1): 2 mg (0.00126 mmol) of an N-hydroxysuccinimide esterderivative of TAMRA (manufactured by GE Healthcare) and 400 μL (1.796mmol) of tetraethoxysilane were mixed.

Step (2): 40 mL of ethanol and 10 mL of 14% aqueous ammonia were mixed.

Step (3): The solution prepared in Step (1) was added to the solutionprepared in Step (2) being stirred at room temperature. The stirring wascontinued for 1 hours from the starting of the addition.

Step (4): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, ethanol was addedto disperse the precipitate therein. The dispersion was centrifugedagain. The precipitate was washed once with each of ethanol and purewater by the same procedure.

The obtained silica nanoparticle b was observed with a scanning electronmicroscope (SEM, model S-800, manufactured by Hitachi, Ltd.). Theaverage particle diameter was 100 nm, and the coefficient of variationof 15%.

Synthesis Example 3 Silica Containing Quantum Dots: Synthesis of SilicaNanoparticles Containing CdSe/ZnS Having an Emission Wavelength of 655nm

“Nanoparticle c” was prepared by a method of the following Steps (1) to(4):

Step (1): 10 μL of a CdSe/ZnS decane dispersion (Qdot655, InvitrogenCorporation) and 40 μL of tetraethoxysilane were mixed.

Step (2): 4 mL of ethanol and 1 mL of 14% aqueous ammonia were mixed.

Step (3): The solution prepared in Step (1) was added to the solutionprepared in Step (2) being stirred at room temperature. The stirring wascontinued for 1.2 hours from the starting of the addition.

Step (4): The reaction mixture was centrifuged at 10000 CI for 60minutes, and the supernatant was removed. Thereafter, ethanol was addedto disperse the precipitate therein. The dispersion was centrifugedagain. The precipitate was washed once with each of ethanol and purewater by the same procedure.

The obtained silica nanoparticle c was observed with an SEM. The averageparticle diameter was 130 nm, and the coefficient of variation was 13%.

Synthesis Example 4 Silica Containing Quantum Dots: Synthesis of SilicaNanoparticles Containing CdSe/ZnS Having an Emission Wavelength of 585nm

“Nanoparticle d” was prepared by a method of the following Steps (1) to(4):

Step (1): 10 μL of a CdSe/ZnS decane dispersion (Qdot585, InvitrogenCorporation) and 40 μL of tetraethoxysilane were mixed.

Step 2): 4 mL of ethanol and 1 mL of 14% aqueous ammonia were mixed.

Step (3): The solution prepared in Step (1) was added to the solutionprepared in Step (2) being stirred at room temperature. The stirring wascontinued for 12 hours from the starting of the addition.

Step (4): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, ethanol was addedto disperse the precipitate therein. The dispersion was centrifugedagain. The precipitate was washed once with each of ethanol and purewater by the same procedure.

The obtained silica nanoparticle d was observed with an SEM. The averageparticle diameter was 120 nm, and the coefficient of variation was 12%.

Synthesis Example 5 Organic Fluorescence-Containing PolystyreneNanoparticles: Synthesis of Cy5-Containing Polystyrene Nanoparticles

“Nanoparticle e” was prepared by a method of the following steps (1) to(3):

Step (1): 1 mg (0.00126 mmol) of an N-hydroxysuccinimide esterderivative of Cy5 (manufactured by GE Healthcare) was dissolved in 60 μLof dichloromethane and 120 μL of ethanol.

Step (2): The solution prepared in Step (1) was added to 1.5 mL of anaqueous dispersion of polystyrene nanoparticles having surfacefunctional amino groups and a particle diameter of 100 nm (manufacturedby Micromod) being vigorously stirred. The stirring was continued for 12hours from the starting of the addition.

Step (3): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, ethanol was addedto disperse the precipitate therein. The dispersion was centrifugedagain. The precipitate was washed once with each of ethanol and purewater by the same procedure.

The obtained polystyrene nanoparticle e was observed with an SEM. Theaverage particle diameter was 100 nm, and the coefficient of variationwas 5%.

Synthesis Example 6 Organic Fluorescence-Containing PolystyreneNanoparticles: Synthesis of TAMRA-Containing Polystyrene Nanoparticles

“Nanoparticle f” was prepared by a method of the following steps (1) to(3):

Step (1): 2 mg (0.00126 mmol) of an N-hydroxysuccinimide esterderivative of TAMRA (manufactured by GE Healthcare) was dissolved in 60μL of dichloromethane and 120 μL of ethanol.

Step (2): The solution prepared in Step (1) was added to 1.5 mL of anaqueous dispersion of polystyrene nanoparticles having surfacefunctional amino groups and a particle diameter of 100 nm (manufacturedby Micromod) being vigorously stirred. The stirring was continued for 12hours from the starting of the addition.

Step (3): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, ethanol was addedto disperse the precipitate therein. The dispersion was centrifugedagain. The precipitate was washed once with each of ethanol and purewater by the same procedure.

The obtained polystyrene nanoparticle f was observed with an SEM. Theaverage particle diameter was 100 nm, and the coefficient of variationwas 6%.

[Binding of Antibodies to Phosphor-Containing Silica Nanoparticle]

Binding of antibodies to the phosphor-containing silica nanoparticles ato d was performed by the following procedure.

Specifically, binding of antibodies to each of the nanoparticles a and cwas performed through Steps (1) to (9) and (12) to (14) to form“particles A and C”, and binding of antibodies to the nanoparticles band d was performed through Steps (1) to (7), (10), (11), and (15) to(17) to form “particles B and D”.

Step (1): 1 mg of each type of the nanoparticles a to d were dispersedin 5 mL of pure water. 100 μL of aminopropyltriethoxysilane aqueousdispersion was added to each dispersion liquid, followed by stirring atroom temperature for 12 hours.

Step (2): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed.

Step (3): Ethanol was added to disperse the precipitate therein. Thedispersion was centrifuged again. The precipitate was washed once witheach of ethanol and pure water by the same procedure. The obtained aminogroup-modified silica nanoparticles a to d were subjected to FT-IRmeasurement. The absorption due to an amino group was observed, and themodification with the amino group was confirmed.

Step (4): The concentration of each amino group-modified silicananoparticles a to d prepared in Step (3) was adjusted to 3 nM with aphosphate buffer physiological saline solution (PBS) containing 2 mMethylenediaminetetraacetic acid (EDTA).

Step (5): SM(PEG)12(succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester,manufactured by Thermo Scientific Inc.) was mixed with each of thesolutions prepared in Step (4) into a final concentration of 10 mM,followed by reaction for 1 hour.

Step (6): Each reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed.

Step (7): PBS containing 2 mM EDTA was added to disperse the precipitatetherein. Each reaction mixture was centrifuged again. The precipitatewas washed three times by the same procedure. Finally, the precipitatewas re-dispersed in 500 μL of PBS.

Step (8): 100 μg of anti-human ER antibodies were dissolved in 100 μL ofPBS, and 1 M dithiothreitol (DTT) was added thereto, followed byreaction for 30 minutes.

Step (9): Excess DTT was removed from the reaction mixture by a gelfiltration column to obtain a reduced anti-human ER antibody solution.

Step (10): 100 μg of anti-HER2 antibodies were dissolved in 100 μL ofPBS, and 1 M dithiothreitol (DTT) was added thereto, followed byreaction for 30 minutes.

Step (11): Excess DTT was removed from the reaction mixture by a gelfiltration column to obtain a reduced anti-HER2 antibody solution.

Step (12): Particle a or c was used as a starting material. The particledispersion obtained in Step (7) and the reduced anti-human ER antibodysolution obtained in Step (9) were mixed in PBS, followed by reactionfor 1 hour.

Step (13): The reaction was stopped by adding 4 μL of 10 mMmercaptoethanol.

Step (14): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, PBS containing 2mM EDTA was added to disperse the precipitate therein. The reactionmixture was centrifuged again. The precipitate was washed three times bythe same procedure. Finally, the precipitate was re-dispersed in 500 μLof PBS to obtain phosphor-containing silica nanoparticles A and C towhich the anti-human ER antibodies were bound.

Step (15): Particle b or d was used as a starting material. The particledispersion prepared in Step (7) and the reduced anti-HER2 antibodysolution prepared in Step (11) were mixed in PBS, followed by reactionfor 1 hour.

Step (16): The reaction was stopped by adding 4 of 10 mMmercaptoethanol.

Step (17): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, PBS containing 2mM EDTA was added to disperse the precipitate therein. The reactionmixture was centrifuged again. The precipitate was washed three times bythe same procedure. Finally, the precipitate was re-dispersed in 500 μLof PBS to obtain phosphor-containing silica nanoparticles B and D towhich the anti-HER2 antibodies were bound.

[Binding of Antibodies to Phosphor-Containing Polystyrene Nanoparticles]

Bindings of antibodies to the phosphor-containing silica nanoparticles eand f were performed by the following procedures.

Specifically, binding of antibodies to the nanoparticle e was performedthrough Steps (1), (2), and (5) to (7) to form “particle E”, and bindingof antibodies to the nanoparticle f was performed through Steps (3),(4), and (8) to (10) to form “particle F”.

Step (1): 100 μg of anti-human ER antibodies were dissolved in 100 μL ofPBS, and 1 NI dithiothreitol (DTT) was added thereto, followed byreaction for 30 minutes.

Step (2): Excess DTT was removed from the reaction mixture by a gelfiltration column to obtain a reduced anti-human ER antibody solution.

Step (3): 100 μg of anti-HER2 antibodies were dissolved in 100 μL ofPBS, and 1 its dithiothreitol (DTT) was added thereto, followed byreaction for 30 minutes.

Step (4): Excess DTT was removed from the reaction mixture by a gelfiltration column to obtain a reduced anti-HER2 antibody solution.

Step (5): The dispersion of the particle e and the reduced anti-human ERantibody solution prepared in Step (2) were mixed in PBS, followed byreaction for 1 hour.

Step (6): The reaction was stopped by adding 4 μL, of 10 mMmercaptoethanol.

Step (7): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, PBS containing 2mM EDTA was added to disperse the precipitate therein. The reactionmixture was centrifuged again. The precipitate was washed three times bythe same procedure. Finally, the precipitate was re-dispersed in 500 ofPBS to obtain phosphor-containing polystyrene particles E to which theanti-human ER antibodies were bound.

Step (8): The dispersion of the particle f and the reduced anti-humanHER2 antibody solution prepared in Step (4) were mixed in PBS, followedby reaction for 1 hour.

Step (9): The reaction was stopped by adding 4 μL of 10 mercaptoethanol.

Step (10): The reaction mixture was centrifuged at 10000 G for 60minutes, and the supernatant was removed. Thereafter, PBS containing 2mM EDTA was added to disperse the precipitate therein. The dispersionwas centrifuged again. The precipitate was washed three times by thesame procedure. Finally, the precipitate was re-dispersed in 500 μL ofPBS to obtain phosphor-containing polystyrene particles F to which theanti-HER2 antibodies were bound.

[Binding of Antibodies to Phosphor]

For comparison, “dye G” was prepared by binding anti-human ER antibodiesto Cy5, and “dye H” was prepared by binding anti-HER2 antibodies toTAMPA in accordance with the following procedure.

Specifically, dye G was prepared through Steps (1), (2), (5), (6), and(9) to (11), and dye H was prepared through Steps (3), (4), (7), (8),and (12) to (14).

Step (1): 100 μg of anti-human ER antibodies were dissolved in 100 ofPBS, and 1 M dithiothreitol (DTT) was added thereto, followed byreaction for 30 minutes.

Step (2): Excess DTT was removed from the reaction mixture by a gelfiltration column to obtain a reduced anti-human ER antibody solution.

Step (3): 100 μg of anti-HER2 antibodies were dissolved in 100 μL ofPBS, and 1 M dithiothreitol (DTT) was added thereto, followed byreaction for 30 minutes.

Step (4): Excess DTT was removed from the reaction mixture by a gelfiltration column to obtain a reduced anti-HER2 antibody solution.

Step (5): The concentration of 1 mg (0.00126 mmol) of anN-hydroxysuccinimide ester derivative of Cy5 (manufactured by GEHealthcare) was adjusted to 3 nM with a phosphate buffer physiologicalsaline solution (PBS) containing 2 mM ethylenediaminetetraacetic acid(EDTA).

Step (6): SM(PEG)12(succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester,manufactured by Thermo Scientific Inc. Inc.) was mixed with the solutionprepared in Step (5) into a final concentration of 10 mM, followed byreaction for 1 hour.

Step (7): The concentration of 2 mg (0.00126 mmol) of anN-hydroxysuccinimide ester derivative of TAMRA (manufactured by GEHealthcare) was adjusted to 3 nM with a phosphate buffer physiologicalsaline solution (PBS) containing 2 mM ethylenediaminetetraacetic acid(EDTA).

Step (8): SM(PEG)12(succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester,manufactured by Thermo Scientific Inc.) was mixed with the solutionprepared in Step (7) into a final concentration of 10 mM, followed byreaction for 1 hour.

Step (9): The reaction mixture prepared in Step (6) and the reducedanti-human ER antibody solution prepared in Step (2) were mixed in PBS,followed by reaction for 1 hour.

Step (10): The reaction was stopped by adding 4 μL of 10 mMmercaptoethanol.

Step (11): Excess mercaptoethanol was removed by a gel filtration columnto obtain a solution of Cy5 to which the reduced anti-ER2 antibodieswere bound (dye G).

Step (12): The reaction mixture prepared in Step (6) and the reducedanti-HER2 an solution prepared in Step (4) were mixed in PBS, followedby reaction for 1 hour.

Step (13): The reaction was stopped by adding 4 of 10 mMmercaptoethanol.

Step (14): Excess mercaptoethanol was removed by a gel filtration columnto obtain a solution of TAMRA to which the reduced anti-HER2 antibodieswere bound (dye H).

Table 8 shows the characteristics of particles A to F and dyes G and Hprepared by the above-described treatments,

TABLE 8 Particle A B C D Phosphor-- Nanoparticle a b c d containingPhosphor Cy5 TAMRA Qdot655 Qdot585 nanoparticle Composition Silica ofmaterial Biomaterial-recognizing Anti- Anti- Anti- Anti- body human ERHER2 human ER HER2 antibody antibody antibody antibody Particle E F DyeG Dye H Phosphor- Nanoparticle e f — containing Phosphor Cy5 TAMRA CY5TAMRA Nanoparticle Composition Polystylene — of materialBiomaterial-recognizing Anti- Anti- Anti- Anti- body human ER HER2 humanER HER2 antibody antibody antibody antibody

[Evaluation Experiment: (1) Tissue Staining with Particles A to F anddyes G and H]

Human mammary tissue was immunostained with particles A to F and dyes Gand H prepared above.

A tissue array slide (CB-A712) manufactured by Cosmo Bio Co., Ltd wasused for a section to be stained. The ER and HER2 stainingconcentrations were observed in advance by DAB staining to prepare threedifferent lots: (1) high expression levels of both of ER and HER2, (2) ahigh expression level of ER and a low expression level of HER2, and (3)low expression levels of both of ER and HER2, and each lot was stained.

(1): A pathological section was immersed in xylene in a container for 30minutes. Xylene was replaced with new xylene three times during theimmersion.

(2): The pathological section was immersed in ethanol in a container for30 minutes. Ethanol was replaced with new ethanol three times during theimmersion.

(3): The pathological section was immersed in water in a container for30 minutes. Water was replaced with new water three times during theimmersion.

(4): The pathological section was immersed in a 10 mM citrate buffersolution (pH 6.0) for 30 minutes.

(5): The section was subjected to autoclave treatment at 121° C. for 10minutes.

(6): The section after the autoclave treatment was immersed in PBS in acontainer for 30 minutes.

(7): PBS containing 1% BSA was placed on the tissue, and the tissue wasleft to stand for 1 hour.

(8): 10 μL of the phosphor-containing nanoparticles A to which theanti-human ER antibodies were bound diluted to 0.05 nM with PBScontaining 1% BSA was mixed with 10 μL of the phosphor-containingnanoparticle B to which the anti-HER2 antibodies were bound diluted to0.05 nM with PBS containing 1% BSA. The mixture was placed on thetissue, and the tissue was left to stand for 3 hours.

(9): 10 μL of the phosphor-containing nanoparticle C to which theanti-human ER antibodies were bound diluted to 0.05 nM with PBScontaining 1% BSA was mixed with 10 μL of the phosphor-containingnanoparticle B to which the anti-HER2 antibodies were bound diluted to0.05 nM with PBS containing 1% BSA. The mixture was placed on the tissueof a different slide from the slide in step (8), and the tissue was leftto stand for 3 hours.

(10): 10 μL of the phosphor-containing nanoparticle E to which theanti-human ER antibodies were bound diluted to 0.05 nM with PBScontaining 1% BSA was mixed with 10 μL of the phosphor-containingnanoparticle F to which the anti-HER2 antibodies were bound diluted to0.05 nM with PBS containing 1% BSA. The mixture was placed on the tissueof a different slide from the slides in steps (8) and (9), and thetissue was left to stand for 3 hours.

(11): 10 μL of the dye G to which the anti-human ER antibodies werebound diluted to 0.05 nM with PBS containing 1% BSA was mixed with 10 μLof the dye H to which the anti-HER2 antibodies were bound diluted to0.05 nM with PBS containing 1% BSA. The mixture was placed on the tissueof a different slide from the slides in steps (8) to (10), and thetissue was left to stand for 3 hours.

(12): The stained section was immersed in PBS in a container for 30minutes.

(13): Aquatex manufactured by Merck Chemicals was added dropwise to thesection, and a cover glass was placed on the section for sealing.

[Evaluation Experiment: (2) Count of Bright Spots in Tissue Stained withParticles A to F and Dyes G and H]

The stained tissue sections were irradiated with excitation light toemit fluorescence. An image of the tissue section was taken with a DSUconfocal microscope manufactured by Olympus Corporation, and the numberof the bright spots and luminescent brightness were determined usingbright spot-counting software G-count manufactured by G-Angstrom.

Cy5 and Qdot655 were observed at an excitation wavelength of 633 nm anda detection wavelength of 660 nm. TAMRA and Qdot585 were observed at anexcitation wavelength of 543 nm and a detection wavelength of 580 nm.

The number of bright spots was an average of the numbers of bright spotsin 30 cells which were counted for each of eight areas in a tissue arrayslide. Luminescent brightness was an average of the sum of fluorescenceintensities which were determined in the entire visual field for each ofthe eight areas.

Tables 9 and 10 show the measurement results of the number of thecounted bright spots and measurement of luminescent brightness in“Experimental examples 1 to 7” in combinations of the three lots and theparticles A to F and dyes G and H.

TABLE 9 4: COMPARATIVE EXPERIMENTAL EXAMPLE 1: EXAMPLE 2: EXAMPLE 3:EXAMPLE EXAMPLE Lot 1 ER expression: HIGH/HER2 expression: HIGH Stainingreagent Particles Particles Particles Dyes G and H A and B C and D E andF The number of 660 nm 150 130 155 Detection bright spots immposible 580nm 175 190 180 Detection immposible Fluorescent 660 nm 800 750 820Detection strength immposible 580 nm 870 980 950 Detection immposible

TABLE 10 EXPERIMENTAL EXAMPLE 5: EXAMPLE 6: EXAMPLE 7: EXAMPLE Lot 1 2 3ER expression: HIGH/ ER expression: HIGH/ ER expression: LOW/ HER2expression: HIGH HER2 expression: LOW HER2 expression: LOW Stainingreagent Particles A and B The number of 660 nm 150 170 20 bright spots580 nm 175 20 10 Fluorescent 660 nm 800 850 160 strength 580 nm 870 150100

Table 9 shows that in Experimental example 4 where an organicfluorescent dye alone to which the antibodies were bound, thefluorescence intensity was too weak to be distinguished from backgroundlight and did not allow detection of the target biosubstance, whereas inExperimental examples 1 to 3 where phosphor-containing particles wereused, the fluorescence intensity was high to allow easy detection of thetarget biosubstance.

Table 10 shows that in Experimental examples 5 to 7 of sections showingdifferent expression levels of ER2 and HER2, the number of the brightspots and the fluorescence intensity vary depending on the respectiveexpression levels and that expression levels of different biosubstancesin a single section can be measured by using particles containingdifferent phosphor and different biosubstance-recognizing bodies as astaining reagent.

INDUSTRIAL APPLICABILITY

The present invention is suitable for quantitative detection of a smallamount of a biosubstance.

1. A tissue staining method comprising: staining a tissue with astaining reagent comprising a particle holding plural phosphors, whereina biosubstance-recognizing body is bound to the particle.
 2. The tissuestaining method according to claim 1, wherein. the particle holdingplural phosphors contains the plural phosphors therein.
 3. The tissuestaining method according to claim 1, wherein the phosphor is afluorescent dye, a semiconductor particle, or a rare-earth elementparticle.
 4. The tissue staining method according claim 1, wherein theparticle holding the plural phosphors has a particle diameter of 40 to500 nm.
 5. A tissue evaluation method, comprising: staining a tissuesection with a staining reagent comprising a phosphor-holding particlewhich holds plural phosphors, wherein a biosubstance-recognizing body ishound to the phosphor-holding particle; counting the number of brightspots of fluorescence in the stained tissue section; and evaluating anexpression level of a biosubstance corresponding to thebiosubstance-recognizing body in the stained tissue section on the basisof the number of the counted bright spots.
 6. The tissue evaluationmethod according to claim 5, comprising: measuring brightness of eachbright spot in the stained tissue section; determining a brightnessdistribution on the basis of the number of the counted bright spots andthe be of each bright spot; calculating the brightness perphosphor-holding particle on the basis of the brightness distribution;calculating the number of the phosphor-holding particles bound to thestained tissue section on the basis of the sum of the measuredbrightnesses of the bright spots and the calculated brightness perphosphor-holding particle; and evaluating the expression level of thebiosubstance in the stained tissue section on the basis of thecalculated number of the phosphor-holding particles.
 7. A biosubstancedetection method for specifically detecting a biosubstance in apathological section, the method comprising: staining the pathologicalsection with a staining reagent; and detecting the biosubstance in thestained pathological section, wherein in the staining of thepathological section, a first particle holding plural first phosphorswhere a first biosubstance-recognizing body is bound to the firstparticle and second particle holding plural second phosphors having adifferent fluorescence wavelength from a fluorescence wavelength of thefirst phosphor where a second biosubstance-recognizing body differentfrom the first biosubstance-recognizing body is bound to the secondparticle are used as the staining reagent.
 8. The biosubstance detectionmethod according to claim 7, wherein the first biosubstance-recognizingbody is an anti-human ER antibody; and the secondbiosubstance-recognizing body is an anti-HER2 antibody.
 9. Thebiosubstance detection method according to claim 7, wherein the firstphosphor and the second phosphor are selected from organic fluorescentdyes and quantum dots.
 10. The biosubstance detection method accordingto claim 7, wherein in the detecting of the biosubstance, an expressionlevel of the biosubstance is determined by counting the number of brightspots.
 11. The biosubstance detection method according to claim 7,wherein in the detecting of the biosubstance, an expression level of thebiosubstance is determined by measuring luminescent brightness.